PROJECT SUMMARY: LIGAND DISCOVERY PROJECT The development of strategies, tools and infrastructure for the discovery of novel microbial metabolism represents a major challenge to the post-genomic biological community. The functional properties of solute binding proteins (SBPs) make them particularly amenable to large-scale functional annotation, as the first step in a catabolic pathway is frequently the passage of a metabolite across the cellular membrane by SBP- dependent transport machinery. The ability to identify the initial reactant (or a closely related molecule) for a catabolic pathway provides an immediate toe-hold by placing significant constraints on the regions of chemical space which need to be considered and, in conjunction with knowledge of colocalized and coregulated genes, begins to define details of the in vivo biochemical transformations operating within the metabolic pathway. Accordingly, a central goal of this project is to evaluate the wide-spread utility of targeting SBPs and related proteins for initial functional insight. We have implemented a high-throughput differential scanning fluorimetry (DSF) assay for the interrogation of ligands/metabolite libraries carefully constituted for specific subfamilies of SBPs. In parallel, high resolution structural characterization of SBP-ligand complexes will reveal the determinants responsible for ligand recognition, and delineate ?specificity boundaries? required for confidently defining the limits of annotation transfer. The Ligand Discovery Project will specifically examine SBPs involved in carbohydrate and amino acid metabolism and expand this strategy to include the ligand-responsive transcriptional regulators which combine an SBP-like domain with a DNA-binding module. Finally, this strategy will be applied to the microbes that compose the human gut microbiome. Combining experimentally defined SBP ligands and high resolution structural information with genome neighborhood networks (Metabolism Project) and additional insights gleaned from computational approaches (Modeling Project) results in a powerful multidisciplinary strategy for the discovery of new metabolism. When systematically applied to the human gut microbiome, these approaches will result in the discovery of new metabolic pathways important for communication between members of the gut community, communication between the gut microbes and the human host, and in particular will expand our understanding of the impact of microbial metabolism on human health and disease. Most importantly, the strategies and tools described in this application will enable the discovery of novel metabolism by the entire community.